Effect of Dexamethasone on Ciprofibrate-lnduced Cell Proliferation

FUNDAMENTAL AND APPLIED TOXICOLOGY 3 5 , 7 8 - 8 3 (1997)

ARTICLE NO FA962264

Effect of Dexamethasone on Ciprofibrate-lnduced CellProliferation and Peroxisome Proliferation

M. SAMBASIVA RAO AND V. SUBBARAO

Department of Pathology, Veterans Affairs Lakeside Medical Center and Northwestern University Medical School, Chicago, Illinois 60611

Received May I, 1996; accepted October 1, 1996

Effect of Dexamethasone on Ciprofibrate-lnduced Cell Prolifer-ation and Peroxisome Proliferation. RAO, M. S., AND SUBBARAO,V. (1997). Fundam. Appl. Toxicol. 35, 78-83.

Peroxisome proliferators cause liver cell proliferation in additionto other pleiotropic effects such as peroxisome proliferation andinduction of certain peroxisomal and cytosolic enzymes in liver.Since dexamethasone has been shown to inhibit mitogen-inducedliver cell hyperplasia, we examined whether dexamethasone inhib-its only cell proliferation without affecting peroxisome prolifera-tion induced by peroxisome proliferators such as ciprofibrate. Liv-ers of rats fed a diet containing ciprofibrate (0.025%) with orwithout added dexamethasone (0.5 mg or 1 mg/kg diet) for 1week were evaluated for hepatocyte proliferation and peroxisomeproliferation. Dexamethasone administration resulted in abroga-tion of ciprofibrate-induced cell proliferation as shown by bromo-deoxyuridine (BrdU) labeling and mitoses counts. The hepatocyteproliferative index measured after administration of a single doseof BrdU was 18.3 ± 1.1 and 2.3 ± 0.7% (/? < 0.01) in ciprofibrateand ciprofibrate + dexamethasone treated rats, respectively. Withmultiple injections of BrdU (daily injections for 7 days) the prolif-erative index was 225 ± 10 and 183 ± 2% (p < 0.02), respectively,in these two groups. Interestingly, whereas the levels of peroxisomeproliferator-induced M, 80,000 polypeptide and catalase and per-oxisomal bifunctional enzyme, and the corresponding mRNAs andperoxisome volume density were unaffected. These results showthat dexamethasone selectively inhibits only cell proliferationwithout inhibiting the peroxisome proliferation caused by ciprofi-brate. This model should be useful for examining the role of cellproliferation versus oxidative stress in peroxisome proliferator-i n d u c e d hepatOCarcinOgeneSJS. © 1997 Society of Toxicology.

Peroxisome proliferators are a diverse group of syntheticand naturally occurring compounds that induce predictablepleiotropic responses in the liver of rats and mice (Reddyand Lalwani, 1983; Moody 1994). These responses includehepatomegaly, hepatocyte proliferation, and peroxisomeproliferation associated with induction of certain peroxi-somal enzymes and transcriptional activation of genes in-volved in peroxisomal fatty acid /3-oxidation (Reddy andLalwani, 1983; Reddy et al, 1986). The peroxisome prolifer-ative activity of these compounds is shown to be a receptor-mediated process (Green, 1992). In addition, long-term ad-

0272-0590/97 S25.OO 7 8

Copyright © 1997 by the Society of Toxicology.All rights of reproduction in any form reserved.

ministration of peroxisome proliferators causes hepatocellu-lar carcinomas (Reddy et al, 1980; Rao and Reddy, 1987).Since peroxisome proliferators are nongenotoxic, the mecha-nism by which they cause liver tumors remains controversial.The suggested mechanisms include oxidative stress resultingfrom sustained induction of H2O2 generating peroxisomalfatty acyl-CoA (Reddy and Rao, 1989) and increased cellproliferation (Butterworth et al., 1992). To resolve the issueof the primacy of oxidative stress versus cell proliferationin peroxisome proliferator-induced hepatocarcinogenesis, anexperimental model is needed in which the cell proliferativeand peroxisome proliferative properties can be dissociated.

Hepatocyte proliferation is induced either after cell loss(partial hepatectomy or necrosis caused by hepatotoxicagents), referred to as compensatory hyperplasia, or aftermitogen stimulation (lead nitrate, ethylene bromide, etc.),referred to as direct hyperplasia (Fausto and Webber,1994; Ledda-Columbano et al, 1992). It has been shownthat during compensatory hyperplasia there is activationof immediate early genes and production of cytokines andgrowth factors (Fausto and Webber, 1994). The mitogen-induced liver cell hyperplasia appears to be dependentmostly on tumor necrosis factor-a (TNFa) and not otherfactors (Coni et al, 1993; Shinozuka et al, 1994). Theglucocorticoid dexamethasone has been shown to inhibitmitogen-induced liver cell proliferation through abroga-tion of TNFa production (Ledda-Columbano et al, 1994).Since peroxisome proliferators also induce liver cell pro-liferation through a mitogenic effect without causing livercell necrosis, we hypothesized that dexamethasone inhib-its only cell proliferation, and not the peroxisome prolifer-ation induced by ciprofibrate, a potent peroxisome prolif-erator. The results demonstrate that dexamethasone pre-vents cell proliferation in liver but not the peroxisomeproliferation induced by ciprofibrate in the rat.

METHODS

Male F-344 rats were obtained from Charles River Laboratories (Wilmin-gton, MA) and housed in plastic cages on San-i-cel bedding in an air-conditioned room with a 12-hr dark and light cycle Aftera week of acclima-tization, rats were divided into six groups (4 to 8 rats/group) and fed a

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DEXAMETHASONE AND LIVER CELL PROLIFERATION 79

diet containing ciprofibrate (0.025%; Sterling-Winthrop Research Institute,Rensselaer, NY) and/or dexamethasone (Sigma) for 7 days as follows:group 1, control diet; group 2, ciprofibrate; group 3. ciprofibrate + dexa-methasone (1 mg/kg diet); group 4. ciprofibrate + dexamethasone (0.5 mg/kg diet); group 5, dexamethasone (1 mg/kg diet); and group 6, dexametha-sone (0.5 mg/kg diet). At the beginning of experiments rats weighed between80 and 90 g. At the end of 7 days all animals were given bromodeoxyuridine(BrdU) intraperitoneally (100 mg/kg body weight) 1 hr prior to euthaniza-tion. In addition, 16 rats of the same age and weight were divided intofour equal groups and fed a diet containing ciprofibrate, ciprofibrate +dexamethasone (0.5 mg/kg diet), dexamethasone (0.5 mg/kg diet), and con-trol diet for 7 days. These animals were given daily ip injections of BrdU(100 mg/kg body weight), a total of seven doses, and euthanized 1 hr afterthe last injection. At the time of euthanization body weights and liverweights were obtained and portions of liver were used for light microscopy,electron microscopy, preparation of postnuclear fraction, and total RNA.Quantitative data were analyzed by Student's r test.

Morphometric analysis of peroxisome. Fragments of liver from fourrats in each group were fixed in 2.5% glutaraldehyde in 0.1 M sodiumcacodylate buffer, pH 7.4, for 4 hr and processed for transmission electronmicroscopy. Thin sections were cut on an LKB ultratome. stained withuranyl acetate and lead citrate, and examined in a JOEL JEM-100CS 11electron microscope. Morphometric analysis of peroxisomes was carriedout as described before (Rao et al, 1982), according to the procedure ofWeibel et al. (1966). Briefly, micrographs were taken at X5000 and magni-fied 2.5 times at printing. Points of intersection overlying cytoplasm andperoxisomes were counted using a 5-mm-spaced lattice grid. The volumedensity of peroxisomes was determined in relation to cytoplasmic volume.Ten photographs were counted from each animal.

Analysis of proliferative indices. For BrdU immunohistochemistry,portions of liver from all animals were fixed in 70% alcohol overnight at4°C and processed for light microscopy. Five-micrometer-thick paraffinsections were stained immunohistochemically by the avidin-biotin-peroxi-dase complex method using Vectastain ABC reagent (Vector Laboratories,Burlingame, CA) with monoclonal antibodies specific for BrdU (Becton-Dickenson) at a dilution of I in 300 (Ward et al, 1988). The reactionproduct was visualized with the addition of diaminobenzidine tetrahydro-chloride and the slides were counterstained with hematoxylin. The labelingindex was obtained by counting 2000 hepatocytes in each liver.

To obtain a mitotic index 2000 hepatocytes were counted from each liveron hematoxylin and eosin stained sections.

SDS-polyacrylamide gel electrophoresis. Ten percent (W/V) liver ho-mogenates were prepared in ice-cold 0.25 M sucrose containing 10 rriMEDTA (pH 7.5) using a Potter- Elvehjem homogenizer. Postnuclear frac-tions were obtained by centrifuging the homogenate at 700^ for 10 min ina Beckman J-21 centrifuge at 4°C The protein concentration in the homoge-nate was determined by the method of Lowry et al. (1951). Electrophoresiswas performed on 10% SDS-polyacrylamide slab gels according to themethod of Laemmli (1970), using 30 y.% protein (Nemali et al., 1988). Aftercompletion of the electrophoretic run (20 mA/slab), the proteins were fixedby immersing the gels in 10% trichloroacetic acid for 30 min and thenstained with 0.1% Coomassie blue in 50% methanol/10% acetic acid.

Northern blot analysis. Total RNA was extracted from livers using themethod of guanidinium thiocyanate-phenol -chloroform extraction (Chom-czynski and Sacchi, 1987). Electrophoresis of glyoxal-denatured total RNA(30 /ig) in 1% agarose gel and transfer to nitrocellulose filters were per-formed (Heilman et al., 1982). Filters were hybridized with nick-translated32P-labeled catalase and peroxisomal bifunctional enzyme (PBE) cDNA at42°C for 18 hr (Nemali et al, 1988). The filters were washed in 2x SSC/0.1% SDS at room temperature for 2 hr, dried, and exposed to film at-70°C. The relative mass of specific RNAs was measured by densitometricscanning of the autoradiographs.

RESULTS

Body and liver weights, proliferative index of hepatocytes,and peroxisome volume density in rats given ciprofibratediet with or without dexamethasone are presented in Table 1.Body weight gain in control rats and rats fed diet containingciprofibrate and ciprofibrate + dexamethasone (0.5 mg/kgdiet) increased approximately by 52, 34, and 13%, respec-tively, in 7 days, whereas no weight gain was observed inrats that were fed diet containing higher dose of dexametha-sone. As expected, ciprofibrate treatment caused a significantincrease in relative liver weight when compared to that ofcontrols (9.0 g vs 4.7 g/100 g body weight, p < 0.001).Similarly, rats that received both dexamethasone and ci-profibrate also showed a significant increase in absolute andrelative liver weights over those of controls. In rats thatwere fed diet containing only dexamethasone there was noincrease in absolute liver weight, although the relativeweight increased by 36 to 60% due to a relative reductionin body weight gain.

BrdU, a pyrimidine analogue, is incorporated into DNAsynthesizing nuclei and can be easily identified immuno-histochemically using anti-BrdU antibodies. Immunohis-tochemical studies performed after administration of sin-gle or multiple doses of BrdU demonstrated a significantincrease in the number of labeled hepatocytes in rats givenciprofibrate, when compared to controls and rats givenciprofibrate + dexamethasone diet (Figs, la and lb). Thelabeling index of hepatocytes after pulse labeling for 1 hrwas 18.3 and 8.5/2000 in ciprofibrate treated and controlrats, respectively (p < 0.01). Interestingly, in rats givendexamethasone and ciprofibrate or dexamethasone alonethere was a marked decrease in the number of labeledcells. Dexamethasone at 0.5 mg and 1 mg/kg diet doselevels completely prevented ciprofibrate-induced cell pro-liferation. In the dexamethasone-treated groups the label-ing indices ranged from 2 to 5/2000 cells. It is not clearwhy the 0.5 mg dexamethasone dose level caused moreinhibition of cell proliferation than the 1 mg dose level.In rats that were given multiple doses of BrdU the labelingindex was 164,225, 183, and 41/2000 cells in the control,ciprofibrate, and ciprofibrate + dexamethasone groups,respectively. Similarly, the mitotic index was also 5- to12-fold higher in the ciprofibrate group when comparedto the dexamethasone + ciprofibrate group (Table 1).

Ultrastructural analysis of hepatocytes from rats fed dietcontaining ciprofibrate alone or ciprofibrate and dexametha-sone (1 mg/kg diet) showed a marked increase in the numberof peroxisomes (Figs. 2a and 2b). Morphometric analysis inboth the groups revealed a 10-fold increase in the volumedensity of peroxisomes over that of control animals (p <0.001), which received normal chow or chow containingdexamethasone (1 mg/kg diet).

SDS-PAGE analysis of postnuclear fractions prepared


80 RAO AND SUBBARAO

TABLE 1Effect of Dexamethasone on Ciprofibrate-Induced Liver Weight, Peroxisome Volume Density, and Hepatocyte Proliferation"

Group

ControlCiprofibrateCiprofibrate + dexamethasone

(1 mg/kg diet)Ciprofibrate + dexamethasone

(0.5 mg/kg diet)Dexamethasone (1 mg/kg

diet)Dexamethasone (0.5 mg/kg

diet)

Bodyweight

137 ± 3*116 ± 2

84 ± 1

89 + 2

84 ± 1

86 ± 1

Liver weight (g)/100 g body weight

4.7 ± 0.19.6 ± 0.2

9.6 ± 0.2

9.4 + 0.1

7.9 + 0.3

7 3 ± 0.2

Peroxisomevolume density

0.7 ± 0 177 ± 1

6.8 ± 1.1

—

0.7 ± 0 1

—

Labeled nuclei/2000 hepatocytes

BrdU, singledose

8.5 ± 1.5'18.3 ± 1.1

4.7 ± 0.9

2.3 ± 0.7

3 5 ± 0.7

2.0 ± 0.5

BrdU, 7

164 ±225 ±

.—

183 ±

—

41 ±

doses

15'10

2

8

Mitotic index/2000 cells

1.8 ± 0.46.5 ± 1

1.2 ± 0.3

0.5 + 02

0.3 + 0.2

0.3 ± 0.2

" Ciprofibrate (0.025%) diet with or without dexamethasone was fed for 7 days.h Mean + SEM of 8 animals.' Mean + SEM of 4 animals.

from livers of different groups is shown in Fig. 3. In ratstreated with ciprofibrate alone or ciprofibrate and dexameth-asone, a marked increase in the amount of peroxisome prolif-erator-induced M, 80,000 polypeptide (PPA-80) was noted,whereas in control groups the level of this polypeptide wasvery low. Northern blot analysis of total RNA obtained fromlivers of ciprofibrate or ciprofibrate and dexamethasoneshowed a marked increase in the levels of PBE mRNA anda slight increase in catalase mRNA (Figs. 4 and 5). Quantita-tive analysis of PBE and catalase mRNA signals by densi-tometry revealed a 10- to 12-fold and a 1.5-fold increase,respectively, in these groups when compared with the controlgroups. The levels of PPA-80 and mRNAs of PBE and cata-lase in the 0.5 and 1 mg dexamethasone + ciprofibrategroups were comparable.

DISCUSSION

The present study, which was designed to examine theeffect of the glucocorticoid dexamethasone on ciprofibrate-induced peroxisome proliferation and hepatocyte prolifera-tion, clearly demonstrates that dexamethasone inhibits cellproliferation without affecting the peroxisome proliferationand PBE induction caused by ciprofibrate. Peroxisome vol-ume density and the levels of catalase and PBE mRNAs wereidentical in the ciprofibrate group and the dexamethasone +ciprofibrate groups. These findings indicate that dexametha-sone did not inhibit activation of the peroxisome proliferator-activated receptor isoforms implicated in the modulation ofperoxisome proliferator-induced pleiotropic effects. None-theless, the effect of dexamethasone on ciprofibrate-induced

FIG. 1. Immunoperoxidase stain for BrdU in the liver of ciprofibrate-treated (a) and control (b) rat. Several labeled nuclei are seen in ciprofibratetreated rat. Original magnification, X200.



FIG. 2. Electron micrograph of hepatocyte (a) from a rat treated with ciprofibrate and dexamethasone (1 mg/kg diet) showing a marked increase inthe number of peroxisomes (arrows) and (b) from dexamethasone-alone treated rat showing an occasional peroxisome (arrow), x 14,000.

cell proliferation was completely different. Dexamethasoneat both dose levels (0.5 mg and 1 mg/kg diet), as revealedby immunohistochemistry after administration of a singledose and multiple doses of BrdU, completely abrogated thecell proliferation induced by ciprofibrate. In addition, dexa-methasone also suppressed cell proliferation in control rats.

Liver cell proliferation is dependent on a multitude ofregeneration signals derived from hepatic and extrahepaticsources. These signals include hormones, growth factors,and cytokines (Bucher. 1995; Fausto el at, 1995). It appearsthat cytokines and growth factors play a major role duringcompensatory hyperplasia, whereas cytokine TNFa mostly


82 RAO AND SLJBBARAO

M 1 2 3 4 8

FIG. 3. SDS-PAGE of liver postnuclear fraction obtained from control(lanes 1 and 2), dexamethasone (lanes 3 and 4), ciprofibrate and dexametha-sone (lanes 5 and 6), and ciprofibrate (lanes 7 and 8) treated rats. M,molecular weight standard; the arrow indicates the position of PPA-80.

plays a crucial role during mitogen-induced direct hyperpla-sia (Fausto and Webber, 1994; Coni et al., 1993; Shinozukaet al., 1994). TNFa that is produced by Kupffer cells inthe liver, through a paracrine effect, stimulates transcriptionfactor (NF-KB) , leading to priming of hepatocytes (Censueet al., 1991; Fausto and Webber, 1994). In addition, it hasalso been shown that TNFa induces DNA synthesis in cul-tured hepatocytes (Beyer and Theologides, 1993). Dexa-methasone, a known inhibitor of TNFa, was shown to inhibitmitogen-induced liver cell proliferation (Ledda-Columbanoet al., 1994). Remick et al. (1989) have shown that dexa-methasone markedly reduced TNF induction caused by lipo-polysacharide in mice. It is possible that inhibition of ci-profibrate-induced cell proliferation by dexamethasone isalso through inhibition of TNFa. Earlier studies have shownthat peroxisome proliferators cause proliferation of both he-patocytes and Kupffer cells (Ward et al., 1988; Rao, personalobservation). In this study we have noticed a marked de-crease in the number of labeled Kupffer cells in the dexa-methasone and ciprofibrate group when compared to that ofthe ciprofibrate treated group (data not shown).

We have presented an experimental model where perox-

1 2 3 4 5 6 7 8 0 1 0 11 12

FIG. 4. Northern blot analysis of PBE mRNA in livers of controls(lanes 1 -3 ) . dexamethasone (lanes 4-6) , ciprofibrate and dexamethasone(lanes 7-9), and ciprofibrate (lanes 10-12) treated rats. The size of PBEmRNA is 3 kb.

FIG. 5. Northern blot analysis for catalase mRNA in livers of rats fromdifferent groups (lanes same as in Fig. 4). The size of catalase mRNA is2.4 kb.

isome proliferator-induced cell proliferation was selec-tively inhibited without affecting peroxisome proliferationand induction of peroxisomal enzymes. This model shouldserve as an ideal system for investigating the role of oxida-tive stress in peroxisome proliferator-induced hepatocar-cinogenesis.

ACKNOWLEDGMENT

This research was supported by a Veterans Affairs Merit Review Grant.

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Effect of Dexamethasone on Ciprofibrate-lnduced Cell Proliferation